For artificial assisted reproductive technology, a common method for improving the success rate of in vitro fertilization and embryo transplantation is cryopreserving more embryos obtained by vitro fertilization or eggs obtained from female in the −196° C. liquid nitrogen for short-term or long-term selective embryo transplantation. The clinical significancy of vitrification of biological sample is that the cryopreserved embryos or cells will stop development and be preserved for decades in coolant by a long-term preservation in a coolant such as liquid nitrogen, besides, upon rewarming the cryopreserved biological samples, one or more biologically live cells can be recovered.
When the materials are subjected to cryopreservation, the contact of material at room temperature with coolant at extremely low temperature (eg. liquid nitrogen) results in Leidenfrost effect, which causes the formation of vapor blanket to block conducting of heat, thereby decreasing the cooling rate of the desired freezing material. Generally, cryoprotectant is necessarily added to prevent the formation of ice crystals during freezing process of biological samples. However, cryoprotectant of high concentration is toxic to biological samples and will result in cells with low quality after recovery. Therefore, a vitrification technique for quick freezing is developed.
Currently, open-type vitrification carriers, such as open pulled straw, quartz capillary, Cryoloop, or Cryotip, etc., are generally used both domestically and abroad for increasing freezing rate. In 2005, Kuwayama proposed a new method, Cryotop method, that is, dipping the vitrification carrier loaded with a sample into the liquid nitrogen directly to increase cooling rate by minimizing the solution volumes. However, the biological sample was preserved in a non-sterilized condition, resulting the risk of cross contamination and the unpredictable direct toxic effect of liquid nitrogen on cells due to the direct contact of biological sample with liquid nitrogen (Covo A, Domingo J, Perez S, et al. Vitrification: an effective new approach to oocyte banking and preserving fertility in cancer patients [J]. Clin Transl Oncol, 2008, 10(5):268-273; Observation on the effects of closed pulled straw vitrification on naive human embryos cryopreservation, Shandong Medical Journal, Vol 50, N0. 15, 2010). The latest research further indicated that low molecular compounds in liquid nitrogen are toxic to biological samples (Y Panagiotidis, P Vanderzwalmen, Y Prapas, E kasapi, et, al Open versus closed vitrification of blastocysts from an oocyte-donation programme: a prospective randomized study. Reproductive BioMedicine Online, In Press, Uncorrected Proof, March 2013). Patent application No. 200710192245.9 disclosed a sealed vitrification device for preventing the exogenous pathogenic contamination to biological samples. However, although a sealed system for the isolation of coolant is obtained, the operation process of this type of cryopreservation carrier is still complicated since several steps such as thermoplastic sealing, etc., are needed.
Thus, there is an urgent need to develop a sealed freezing device for biological samples with high efficiency of vitrification, easy operation, safe and non-toxicity.